Chromatographic methods for purification of proteins from plasma

ABSTRACT

The present invention relates to the field of chromatography. More closely, the invention relates to a chromatographic method for purification of proteins, such as Factor VIII, von Willebrand factor and Factor IX. The chromatographic method is performed on a matrix comprising an inner porous core and outer porous lid surrounding said core.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/564,232, filed on Oct. 4, 2017, which claims the priority benefit ofPCT/EP2016/057356 filed on Apr. 4, 2016, which claims the prioritybenefit of Great Britain Application No. 1506117.9 filed on Apr. 10,2015. The entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of chromatography. Moreclosely, the invention relates to a chromatographic method forpurification of plasmaproteins, such as Factor VIII, von Willebrandfactor and Factor IX. The chromatographic method is performed on amatrix comprising an inner porous core and outer porous lid surroundingsaid core.

BACKGROUND OF THE INVENTION

Blood contains different types of cells and molecules which arenecessary for vital body functions, and is therefore collected fortherapeutic purposes, eg for blood transfusions. However, it is possibleto separate and prepare different fractions from blood, such as redblood cells or cell-free plasma, which enables a more directedtherapeutic treatment of medical conditions. Several proteins in plasmacan also be further isolated and used for specific therapeutictreatments, eg albumin is used to restore blood volume, immunoglobulinsare used for immune deficiencies, and coagulation factors are used forblood coagulation disorders.

Plasma contains proteins of different function, different size,different amount, etc, so there are different methods for purificationof the different plasma proteins. The purification processes are oftendesigned to obtain several target proteins from one single starting poolof plasma. The processes typically involve precipitation orchromatography steps or a combination thereof. Chromatography is oftenused to increase the purity of the target protein and reduce the riskfor detrimental side effects. Many plasma proteins exhibit very potentactivities, and if present as contaminants, they can cause adversereactions even at very low levels, when administered to patients.

Collected human plasma is stored frozen, and the initial step in aplasma protein purification process is thawing and pooling of plasma.When thawing at low temperatures, typically 1-6 degrees C., some plasmaproteins precipitate and can be collected by eg centrifugation. Thecollected precipitate is called cryoprecipitate, and can be used as asource of eg coagulation Factor VIII (FVIII) and von Willebrand Factor(vWF). Most of the FVIII in plasma is present as a complex with thelarge vWF multimers, and the two proteins are therefore oftenco-purified. The remaining liquid after removal of the cryprecipitate isoften referred to as cryodepleted plasma or cryosupernatant, and thiscan be used as a source of eg albumin, immunoglobulin G (IgG),coagulation Factor IX (FIX).

The purification of many plasma proteins can be challenging. This candepend on the presence of small amounts of contaminants with undesiredbut potent activity, or that the proteins sometimes lose their activityor gain unwanted activity. For example, the FVIII easily loses activity,and the known methods used for purification are not satisfactory in manyrespects. Thus, there is a need of improved methods which can beoperated at conditions where the proteins retain their activity, inorder to obtain plasma products in good yields.

SUMMARY OF THE INVENTION

The present invention provides chromatographic materials and methods forpurification of plasma proteins, especially for human plasmaapplications. The chromatographic materials are capable of separationsbased on different principles, eg size and bind/elute. The method of theinvention is especially suitable for purification of plasma sampleswhich contain both large proteins (eg FVIII/vWF) and smaller proteins(HSA, IgG, FIX) of therapeutic interest.

Thus, the present invention provides a chromatographic method forpurification of therapeutic proteins from plasma, comprising thefollowing steps: loading plasma on a chromatography column packed with aresin comprising porous shell beads having an inner porous core and anouter porous shell, wherein the inner core is provided with anionexchange ligands and the shell is inactive (ie not provided with anyligands) and wherein the porosity of the lid and core does not allowentering of molecules larger than 500 kD, such as FIII/vWF; adsorbingFactor IX (FIX) on the anion exchange ligands in the core; collectingseparated plasma proteins in the flow through; and eluting FIX from theligands in the core. Adjustments of running and elution buffer in achromatographic method are within the knowledge of the skilled person inthe field and examples thereof are described in the experimental sectionbelow.

The anion exchange ligands may be any anion exchange ligands but arepreferably selected from diethylaminoethyl (DEAF), quaternary aminoethyl(QAE) or quaternary ammonium (Q), most preferably the anion exchangeligands are Q-ligands.

In a preferred embodiment other plasma proteins besides FIX arecollected in the flow through separated from each other by the sievingeffect of the core and shell and comprise Factor VIII (FVIII) and vonWillebrand factor (vWF), IgG, human serum albumin (HSA) and ComplementC3. See the chromatogram in FIG. 1B below, describing peaks A, B and C.

In an alternative embodiment the loading of plasma is repeated 1-20times, preferably 5-10 times, followed by running buffer to obtain acorresponding number of flow throughs (FT's), before the FIX is elutedfrom the ligands in the core. Each loading is followed by running bufferof about one column volume and a corresponding number of FT's as numberof loadings are obtained. This is described more closely in FIGS. 1A-1Bbelow.

In the alternative embodiment specific fractions of respective FT arepooled to obtain FVIII/vWF, IgG/HSA and C3 respectively. Thus, on oneand the same chromatography column separated plasma fractions areobtained for FVIII/vWF, IgG/HSA, C3 and FIX.

Preferably the total shell bead (i.e. shell plus core) thickness is40-100 μm in diameter, and the lid thickness is preferably 2-10 μm. Theligand concentration in the core is preferably 50-200 μmole/ml.

In a further embodiment the shell is provided with affinity ligands,hydrophobic interaction ligands, IMAC ligands, cation exchange ligandsor multimodal ligands (or any ligands that reply on another separationprinciple than anion exchange ligands); and other plasma protein thanFIX, such as FVIII/vWF, IgG, HSA, C3, are adsorbed on the ligands in theshell in the same step as FIX is adsorbed on the ligands in the core;and wherein plasma proteins are sequentially eluted from the ligands inthe shell (FVIII/vWF and/or IgG, HSA, C3, depending on chosen type ofligand) and ligands in the core (FIX).

Preferably the ligands in the shell are ligands having immunoglobulinaffinity, such as Protein A or G or variants thereof known in the art.

The porous material used in the method of the invention is preferably asieving material, such as a gel filtration material commonly used forchromatography. The porous material in the inner core may have the sameor different porosity as the porous material in the lid. The porousmaterial is derived from a synthetic polymer material, such as styreneor styrene derivatives, divinylbenzene, acrylamides, acrylate esters,methacrylate esters, vinyl esters and vinylamides, or from a naturalpolymer material, such as carbohydrate material selected from agarose,agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan andalginate. The preferred porous material is agarose.

The shell and core may be made of agarose of the same porosity but theymay also be made of different porosity.

In a second aspect the invention relates to use of plasma protein(s)obtained from the above method for therapy. For example albumin is usedto restore blood volume, immunoglobulins are used for immunedeficiencies, and coagulation factors are used for blood coagulationdisorders.

It is contemplated that the plasma proteins, especially FIX, may be usedfor therapy without further purification from contaminants, but withnecessary adjustments for biocompatibility etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Chromatogram of plasma applied to prototype 99 column. Numbers1-6=flow through (FT) peaks from the 6 sample applications; E=elutionpeak; CIP=cleaning-in-place.

Absorbance 280 nm—solid line; conductivity—dotted line; pH—dashed line.

FIG. 1B: Partial enlargement of chromatogram of plasma applied toprototype 99 column according to FIG. 1A, showing final (6th) sampleapplication and elution. 6=flow through peaks from the 6^(th) sampleapplication; A=large molecules, eg FVIII/vWF; B=smaller molecules, egalbumin, IgG; C=partially retained material; E=eluted molecules, eg FIX.

Absorbance 280 nm—solid line; conductivity—dotted line; pH—dashed line.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described more closely in connection with somenon-limiting examples and the accompanying figures.

Example 1: Synthesis of Prototypes

General

Volumes of matrix refer to settled bed volume and weights of matrixgiven in gram refer to suction dry weight. For large scale reactionstirring is referring to a suspended, motor-driven stirrer since the useof magnet bar stirrer is prompt to damage the beads. Conventionalmethods were used for the analysis of the functionality and thedetermination of the degree of allylation, or the degree of ligandcontent on the beads.

Support Particles

The support particles used were highly crosslinked agarose beads,prepared according to the methods described in U.S. Pat. No. 6,602,990,which is hereby incorporated by reference in its entirety. The beads hada volume-weighted average diameter (D50,v) of 88 micrometers and a poresize distribution such that 69% of the pore volume was available todextran molecules of Mw 110 kDa. This can also be expressed such that Kdfor dextran 110 kDa on the beads was 0.69, when measured according tothe methods described in “Handbook of Process Chromatography, A Guide toOptimization, Scale-Up and validation” (1997) Academic Press, San Diego.Gail Sofer & Lars Hagel eds. ISBN 0-12-654266-X, p. 368.

Allylation (Prototype 76)

250 mL (g) of support particles were washed 6× gel volumes (GV) withdistilled water, and then 3×GV with 50% NaOH. The gel was then suckeddry and transferred to a 2 L round bottom flask. 485 mL of 50% NaOH wasadded, mechanical propeller stirring was applied and the flask wasimmersed into a water bath at 50° C. After 30 minutes 80 mL of allylglycidyl ether (AGE) was added. The reaction progressed for 18.5 h. Thegel was washed 1×GV with distilled water, 3×GV with ethanol and then8×GV with distilled water.

The allyl content, 276 μmol/mL, was measured by titration.

Prototype 99

Partial Bromination and Shell Inactivation

171.8 g of allylated gel slurry (prototype 76) was transferred to aglass filter (por. 2) and sucked dry. The dry gel is transferred to a1000 mL round bottom flask fitted with a mechanical stirrer. 571 g ofdistilled water is added and the suspension is stirred at 300 rpm. 83.7g of the 1.6% bromine solution is added during 1.5 min. After theaddition the suspension is still white. The reaction proceeded for 15min at rt. The round bottom flask was immersed in a bath and when thetemperature had reached 50° C., 52 g of 50% NaOH was added. The reactionwas let to stand for 17 hours. The reaction is transferred to a glassfilter (por. 2) and washed with distilled water 10×1 GV. The remainingallyl content, 216 μmol/mL, was measured by titration. This correspondsto a theoretical shell thickness of 3.5 μm.

Core Bromination and Q Coupling

41.5 mL (g) of partial allylated base matrix from above was transferreddrained to a 250 mL round bottom flask fitted with a mechanical stirrer.10.37 g of distilled water and 1.66 g of sodium acetate was added. Afterstirring for a couple of minutes, 0.66 mL bromine is added with apipette and the reaction is stirred at 300 rpm for additionally 20 min.Excess bromine is consumed by adding 4.15 mL of 40% sodium formatesolution. The reaction is colourless. After 15 min, 8.30 mL of trimethylammonium chloride (TMAC) is added and the pH is adjusted to 11-11.5 byadding 50% NaOH. The reaction is stirred at 250 rpm at 30° C. for 18 h.The reaction is neutralized by adding 60% acetic acid to pH 5-7 beforetransferring to a glass filter (por. 3). The gel was washed withdistilled water 10×1 GV, followed by 20% EtOH 2×1 GV. Titration of theion exchange groups gave a Q ligand density of 125 μmol/ml.

Table 1 shows the lid thickness, ligand type and concentration inPrototype 99.

TABLE 1 Lid thickness Lid ligand Core ligand Resin (μm) (type, conc.)(type, conc.) Prototype 3.5 No ligand Q, 125 99 μmol/ml

Example 2: Chromatography of Plasma on Prototype 99

Sample

The sample was human plasma. Frozen human plasma was thawed and filteredthrough cotton, and applied to the column.

Buffers and Running Conditions

Column: Tricorn 10/300 with prototype 99, bed height 28.6 cm, columnvolume (CV) 22.5 mL.

Chromatography: Sample volume 6×5.2 mL (6×0.23 CV). Flow rate 50 cm/h(0.65 mL/min).

Running buffer: 20 mM Na-citrate, 0.15 M NaCl, 2.6 mM CaCl₂), pH 7.0.

Elution buffer: 20 mM Na-citrate, 0.5 M NaCl, 2.6 mM CaCl₂, pH 7.0.

Cleaning-in-place (CIP): 0.5 M NaOH.

The column was equilibrated with running buffer prior to the firstsample application. 0.23 CV of plasma was applied to the column,followed by 1.8 CV of running buffer. This procedure, application of0.23 CV of plasma followed by 1.8 CV of running buffer, was repeated 5times, resulting in a total of 6 plasma sample applications. After thefinal 1.8 CV of running buffer, the column was eluted with 1.5 CV ofhigh salt elution buffer. The column was then subjected to CIP byapplying 1.5 CV of 0.5 M NaOH. Finally, the column was re-equilibratedby 4 CV of running buffer.

Analysis

Selected fractions were analyzed for FVIII activity (ChromogenixCoamatic Factor VIII kit), vWF (Technozym vWF:Ag ELISA kit), FIX (ROXFactor IX kit), and by SDS PAGE, and liquid chromatography-massspectrometry (LC-MS).

The prototype 99 was packed in a Tricorn 10/300 column. The bed heightwas 28.6 cm (CV 22.5 mL) and the flow rate was 50 cm/h, to enablesize-dependent group separation. The run consisted of 6 plasma sampleapplications (6×0.23 CV) resulting in 6 group separations, and one finalhigh salt elution from the Q ligand in the core. The fractions from the6 group separations were pooled so that one pooled fraction A with verylarge molecules, one pooled fraction B with smaller proteins, and onepooled fraction C with slightly retained smaller proteins, wereobtained. The high salt elution resulted in one eluted fraction E. Allfractions were analysed for FVIII, vWF and FIX. The chromatogram isshown in FIG. 1A and a partial enlargement is shown in FIG. 1B.

See Table 2 below for analytical results and yield calculations. Theyields of FVIII and vWF in the fraction A pool was 37% and 40%,respectively. Fraction B and C pools had FVIII and vWF activities belowlevel of quantification. The eluted fraction E contained 22% of theFVIII activity, and 13% of the vWF activity. This indicated that most ofthe large FVIII/vWF complexes do not enter the beads, and pass in theflow through, separated from the smaller molecules such as albumin andIgG in fraction B, which enter the beads but do not bind to the Q ligandin the core under these conditions. SDS PAGE and LC-MS showed thatalbumin and IgG were the main proteins in fraction B. LS-MC indicatedthat the major components in Fraction C were C3 and albumin. However,some FVIII/vWF complexes enter the pores and bind to the Q ligand, thiscould depend on the varying size of the FVIII/vWF complexes and the lowflow rate. Fraction E consisted of molecules eluted with high salt, andit was the only fraction with FIX activity, 91% yield. This shows thatthe smaller FIX molecules enter the chromatography beads and bind to theQ ligand in the core. This demonstrates that by using a chromatographymedia with an inactive lid and ligand-containing core, it is possible toseparate large (FVIII/vWF) and small (FIX) plasma proteins which bothbind to the core ligand, as the large proteins are collected in the flowthrough, and the smaller molecules can be collected by elution.

TABLE 2 Plasma on prototype 99. FVIII, vWF and FIX activity. Forfractions, see FIG. 1 A and B. X = not measured. LOQ = level ofquantification (FVIII: 8 mU/mL, vWF: 0.10 U/mL, FIX: 30 mU/mL). VolFVIII FVIII FVIII vWF vWF vWF FIX FIX FIX Sample (mL) (mU/mL) (mU) (%)(U/mL) (U) (%) (mU/mL) (mU) (%) Plasma 31.0 875 27125 100 1.00 31.0 1001360 42160 100 Fraction A 35.7 280 9996 37 0.35 12.5 40 <LOQ Fraction B66.6 <LOQ <LOQ <LOQ Fraction C 47.3 <LOQ <LOQ <LOQ Fraction E 8.2 7436093 22 0.49 4.0 13 4673 38319 91

The invention claimed is:
 1. A chromatographic method comprising thefollowing steps: loading proteins on a chromatography column packed witha resin comprising porous lid beads having an inner porous core and anouter porous lid, wherein the inner core is provided with anion exchangeligands, and wherein the porosity of the lid and core does not allowentering of molecules larger than 500 kD; adsorbing Factor IX (FIX) onthe anion exchange ligands in the core; collecting separated proteins inthe flow through; and eluting FIX from the ligands in the core.
 2. Themethod of claim 1, wherein the anion exchange ligands are selected fromdiethylaminoethyl (DEAE), quaternary aminoethyl (QAE) or quaternaryammonium (Q).
 3. The method of claim 2, wherein the anion exchangeligands are Q-ligands.
 4. The method of claim 1, wherein other proteinsbesides FIX are collected in the flow through separated from each otherby the sieving effect of the core and shell and comprise Factor VIII(FVII) and von Willebrand factor (vWF), IgG, human serum albumin (HSA)and Complement C3 (C3).
 5. The method of claim 1, wherein the loading ofproteins is repeated 1-20 times, followed by running buffer to obtain acorresponding number of flow throughs (FT's), before the FIX is elutedfrom the ligands in the core.
 6. The method of claim 5, wherein otherproteins besides FIX are collected in the flow through separated fromeach other by the sieving effect of the core and shell and compriseFactor VIII (FVIII) and von Willebrand Factor (vWF), IgG, human serumalbumin (HSA) and Complement C3 (C3), and wherein specific fractions ofrespective FT are pooled to obtain FVIII/vWF, IgG/HSA and C3respectively.
 7. The method of claim 1, wherein the total lid beadthickness is 40-100 μm in diameter, and the lid thickness is 2-10 μm. 8.The method of claim 1, wherein the ligand concentration in the core is50-200 μmol/ml.
 9. The method of claim 1, wherein the lid is providedwith affinity ligands, hydrophobic interaction ligands, IMAC ligands,cation exchange ligands or multimodal ligands; and at least one proteinother than FIX are adsorbed on the ligands in the lid in the same stepas FIX is adsorbed on the ligands in the core; and wherein proteins aresequentially eluted from the ligands in the lid and FIX is eluted fromligands in the core.
 10. The method of claim 9, wherein the ligands inthe lid are ligands having immunoglobulin affinity.
 11. The method ofclaim 1, wherein the lid and core are made of agarose of the sameporosity.
 12. The method of claim 1, wherein the porosity of the lid islarger than of the core.
 13. The method of claim 1, wherein the porosityof the lid is smaller than of the core.
 14. The method of claim 5,wherein the loading of proteins is repeated 5-10 times.
 15. The methodof claim 9, wherein the at least one protein other than FIX compriseFVIII/vWF, IgG, HSA, or C3.
 16. The method of claim 1, wherein the lidis provided with ligands comprising at least one of Protein A or G orvariants thereof.